Erythro-VLPs: Anchoring SARS-CoV-2 spike proteins in erythrocyte liposomes.

Novel therapeutic strategies are needed to control the SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) pandemic. Here, we present a protocol to anchor the SARS-CoV-2 spike (S-)protein in the cytoplasmic membranes of erythrocyte liposomes. A surfactant was used to stabilize the S-protein's structure in the aqueous environment before insertion and to facilitate reconstitution of the S-proteins in the erythrocyte membranes. The insertion process was studied using coarse grained Molecular Dynamics (MD) simulations. Liposome formation and S-protein anchoring was studied by dynamic light scattering (DLS), ELV-protein co-sedimentation assays, fluorescent microcopy and cryo-TEM. The Erythro-VLPs (erythrocyte based virus like particles) have a well defined size of ∼200 nm and an average protein density on the outer membrane of up to ∼300 proteins/µm2. The correct insertion and functional conformation of the S-proteins was verified by dose-dependent binding to ACE-2 (angiotensin converting enzyme 2) in biolayer interferometry (BLI) assays. Seroconversion was observed in a pilot mouse trial after 14 days when administered intravenously, based on enzyme-linked immunosorbent assays (ELISA). This red blood cell based platform can open novel possibilities for therapeutics for the coronavirus disease (COVID-19) including variants, and other viruses in the future.

Erythrocytes: Central Actors in Multiple Scenes of Atherosclerosis.

The development and progression of atherosclerosis (ATH) involves lipid accumulation, oxidative stress and both vascular and blood cell dysfunction. Erythrocytes, the main circulating cells in the body, exert determinant roles in the gas transport between tissues. Erythrocytes have long been considered as simple bystanders in cardiovascular diseases, including ATH. This review highlights recent knowledge concerning the role of erythrocytes being more than just passive gas carriers, as potent contributors to atherosclerotic plaque progression. Erythrocyte physiology and ATH pathology is first described. Then, a specific chapter delineates the numerous links between erythrocytes and atherogenesis. In particular, we discuss the impact of extravasated erythrocytes in plaque iron homeostasis with potential pathological consequences. Hyperglycaemia is recognised as a significant aggravating contributor to the development of ATH. Then, a special focus is made on glycoxidative modifications of erythrocytes and their role in ATH. This chapter includes recent data proposing glycoxidised erythrocytes as putative contributors to enhanced atherothrombosis in diabetic patients.

A Profound Membrane Reorganization Defines Susceptibility of Plasmodium falciparum Infected Red Blood Cells to Lysis by Granulysin and Perforin.

Malaria remains one of the most serious health problems in developing countries. The causative agent of malaria, Plasmodium spp., have a complex life cycle involving multiple developmental stages as well as different morphological, biochemical and metabolic requirements. We recently found that γδ T cells control parasite growth using pore-forming proteins to deliver their cytotoxic proteases, the granzymes, into blood residing parasites. Here, we follow up on the molecular mechanisms of parasite growth inhibition by human pore-forming proteins. We confirm that Plasmodium falciparum infection efficiently depletes the red blood cells of cholesterol, which renders the parasite surrounding membranes susceptible to lysis by prokaryotic membrane disrupting proteins, such as lymphocytic granulysin or the human cathelicidin LL-37. Interestingly, not the cholesterol depletion but rather the simultaneous exposure of phosphatidylserine, a negatively charged phospholipid, triggers resistance of late stage parasitized red blood cells towards the eukaryotic pore forming protein perforin. Overall, by revealing the molecular events we establish here a pathogen-host interaction that involves host cell membrane remodeling that defines the susceptibility towards cytolytic molecules.

Erythrocyte membrane proteins involved in the immune response to Plasmodium falciparum and Plasmodium vivax infection.

Invasion of Plasmodium into the red blood cell involves the interactions of a substantial number of proteins, with red cell membrane proteins as the most involved throughout the process from entry to exit. The objective of this work was to identify proteins of the human erythrocyte membrane capable of generating an antigenic response to P. falciparum and P. vivax infection, with the goal of searching for new molecular targets of interest with an immunological origin to prevent Plasmodium infection. To identify these proteins, an immunoproteomic technique was carried out in four stages: protein separation (electrophoresis), detection of antigenic proteins (western blotting), identification of proteins of interest (mass spectrometry), and interpretation of the data (bioinformatic analysis). Four proteins were identified from extracts of membrane proteins from erythrocytes infected with P. falciparum: Spectrin, Ankyrin-1, Band 3 and band 4.2, and a single protein was identified from erythrocytes infected with P. vivax: Band 3. These results demonstrate that modifications in the red blood cell membrane during infection with P. falciparum and P. vivax can generate an immune response, altering proteins of great structural and functional importance.

Case Report: First Case of Cefotaxime-Sulbactam-Induced Acute Intravascular Hemolysis in a Newborn With ABO Blood Type Incompatibility by the Mechanism of Non-Immunologic Protein Adsorption.

Background: ABO blood type incompatibility hemolytic disease of newborn (ABO-HDN) and drug-induced immune hemolytic anemia (DIIHA) due to non-immunologic protein adsorption (NIPA) mainly cause extravascular hemolysis. All the reported severe DIIHA were caused by drug-induced antibodies, and rare report of acute intravascular hemolysis was caused by the NIPA mechanism or ABO-HDN. Case presentation: We report the first case of acute intravascular hemolysis induced by cefotaxime sodium - sulbactam sodium (CTX - SBT) in a case of ABO-HDN which resulted in death at 55 h after birth. The mother's blood type was O and RhD-positive, and the newborn's blood type was B and RhD-positive. No irregular red blood cell (RBC) antibodies or drug-dependent antibodies related to CTX or SBT was detected in the mother's plasma and the plasma or the RBC acid eluent of the newborn. Before the newborn received CTX - SBT treatment, the result of direct antiglobulin test (DAT) was negative while anti-B was positive (2 +) in both plasma and acid eluent. After the newborn received CTX - SBT treatment, the results of DAT for anti-IgG and anti-C3d were both positive, while anti-B was not detected in plasma, but stronger anti-B (3 +) was detected in acid eluent. In vitro experiments confirmed that NIPA of SBT promoted the specific binding of maternal-derived IgG anti-B to B antigen on RBCs of the newborn, thereby inducing acute intravascular hemolysis. Conclusion: The NIPA effect of SBT promoted the specific binding of mother-derived IgG anti-B in newborn's plasma to the newborn's RBC B antigens and formed an immune complex, and then activated complement, which led to acute intravascular hemolysis. Drugs such as SBT with NIPA effect should not be used for newborns with HDN.

Chemo-immunotherapy with doxorubicin prodrug and erythrocyte membrane-enveloped polymer nano-vaccine enhances antitumor activity.

There are plenty of evidences to show that combining of chemotherapy and immunotherapy should be a very potent anti-cancer therapeutic method. In this research, we evaluated the anti-tumor activity of doxorubicin prodrug combined with erythrocyte membrane-enveloped polymer nano-vaccine against tumor in vitro and in vivo. We also analyzed the immune cell populations to investigate the effect in the tumor microenvironment and explored the inhibitory of lung metastasis. Our study showed that chemo-immunotherapy could inhibit the growth of melanoma tumor in mice. This combination approach promoted a stronger immune response by up-regulating the expression of dendritic cells and cytotoxic T cells in lymph nodes and increased the secretion of cytokines. It also restricted the immunosuppressive environment through inhibiting expression of regulatory T cells. It suggested that combination of prodrug and nano-vaccine achieved better anti-tumor effect than monotherapy, which should be widely valued and further studied to establish in a more economical and efficient way, expected to apply in the future clinical practice of cancer treatment.

Band 3 ectopic expression in colorectal cancer induces an increase in erythrocyte membrane-bound IgG and may cause immune-related anemia.

Autoimmune hemolytic anemia (AIHA) is a rare comorbidity in colorectal cancer (CRC) and has an unknown etiology. Previously, we described an AIHA case secondary to CRC with ectopic band 3 expression. Herein, we investigated ectopic band 3 expression and erythrocyte membrane-bound IgG in a CRC cohort. Between September 2016 and August 2018, 50 patients with CRC and 26 healthy controls were enrolled in the present study. The expression of band 3 and SLC4A1 mRNA was observed in 97% of CRC surgical specimens. Although clinical AIHA was not observed in any patient with CRC, a direct antiglobulin test was positive in 10 of the patients in the CRC group (p = 0.01). Flow cytometry revealed significantly increased erythrocyte membrane-bound IgG among patients with CRC compared to healthy controls (mean ± standard deviation; 38.8 ± 4.7 vs. 29.9 ± 15.6, p = 0.012). Normocytic anemia was observed, including in cases negative for fecal occult blood, suggesting a shortened erythrocyte life-span due to increased membrane-bound IgG. Immunoprecipitation revealed increased anti-band 3 autoantibodies in patients' sera. Mouse experiments recapitulated this phenomenon. We also confirmed that band 3 expression is controlled by 5'AMP-activated protein kinase under hypoxic conditions. These findings increase our understanding of the etiology of cancer-related anemia.

Vaccination accelerates hepatic erythroblastosis induced by blood-stage malaria.

BACKGROUND: Vaccination induces survival of otherwise lethal blood-stage infections of the experimental malaria Plasmodium chabaudi. Blood-stage malaria induces extramedullary erythropoiesis in the liver. This study investigates how vaccination affects the course of malaria-induced expression of erythrocytic genes in the liver. METHODS: Female Balb/c mice were vaccinated at week 3 and week 1 before challenging with 106 P. chabaudi-parasitized erythrocytes. The non-infectious vaccine consisted of erythrocyte ghosts isolated from P. chabaudi-infected erythrocytes. Gene expression microarrays and quantitative real-time PCR were used to compare mRNA expression of different erythrocytic genes in the liver of vaccination-protected and non-protected mice during infections on days 0, 1, 4, 8, and 11 p.i. RESULTS: Global transcriptomics analyses reveal vaccination-induced modifications of malaria-induced increases in hepatic gene expression on days 4 and 11 p.i. On these days, vaccination also alters hepatic expression of the erythropoiesis-involved genes Ermap, Kel, Rhd, Rhag, Slc4a1, Gypa, Add2, Ank1, Epb4.1, Epb4.2, Epb4.9, Spta1, Sptb, Tmod1, Ahsp, Acyp1, Gata1, Gfi1b, Tal1, Klf1, Epor, and Cldn13. In vaccination-protected mice, expression of these genes, except Epb4.1, is significantly higher on day 4 p.i. than in un-protected non-vaccinated mice, reaches maximal expression at peak parasitaemia on day 8 p.i., and is slowed down or even decreased towards the end of crisis phase on day 11 p.i.. After day 1 p.i., Epor expression takes about the same course as that of the other erythroid genes. Hepatic expression of Epo, however, is delayed in both vaccinated and non-vaccinated mice for the first 4 days p.i. and is maximal at significantly higher levels in vaccinated mice on day 8 p.i., before declining towards the end of crisis phase on day 11 p.i. CONCLUSION: The present data indicate that vaccination accelerates malaria-induced erythroblastosis in the liver for 1-2 days. This may contribute to earlier replenishment of peripheral red blood cells by liver-derived reticulocytes, which may favour final survival of otherwise lethal blood-stage malaria, since reticulocytes are not preferred as host cells by P. chabaudi.

Detergent-free isolation of native red blood cell membrane complexes.

Over the past few decades, studies on the red blood cell (RBC) membrane gave rise to increasingly sophisticated although divergent models of its structural organization, since investigations were often performed in denaturing conditions using detergents. To access soluble isolated RBC membrane complexes with the preservation of their interactions and conformations, we decided to apply the recent SMALP (Styrene Maleic Acid Lipid Particles) technology to RBC ghosts. Depending on the ionic strength of buffers in which ghost membranes were resuspended, the isolated proteins within SMALPs could differ on Coomassie-stained gels, but with few changes when compared to ghost membrane SDS lysates. We subsequently produced SMALPs derived from ghosts from two different blood group phenotypes, RhD-positive and RhD-negative, both types of RBC expressing the RhCE proteins but only RhD-positive cells being able to express the RhD proteins. This allowed the isolation, by size exclusion chromatography (SEC), of soluble fractions containing the Rh complex, including the RhD protein or not, within SMALPs. The use a conformation-dependent anti-RhD antibody in immunoprecipitation studies performed on SEC fractions of SMALPs containing Rh proteins clearly demonstrated that the RhD protein, which was only present in SMALPs prepared from RhD-positive RBC ghosts, has preserved at least one important conformational RhD epitope. This approach opens new perspectives in the field of the erythroid membrane study, such as visualization of RBC membrane complexes in native conditions by cryo-electron microscopy (CryoEM) or immuno-tests with conformation-dependent antibodies against blood group antigens on separated and characterized SMALPs containing RBC membrane proteins.

Antigenic cartography of immune responses to Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1).

Naturally acquired clinical immunity to Plasmodium falciparum is partly mediated by antibodies directed at parasite-derived antigens expressed on the surface of red blood cells which mediate disease and are extremely diverse. Unlike children, adults recognize a broad range of variant surface antigens (VSAs) and are protected from severe disease. Though crucial to the design and feasibility of an effective malaria vaccine, it is not yet known whether immunity arises through cumulative exposure to each of many antigenic types, cross-reactivity between antigenic types, or some other mechanism. In this study, we measured plasma antibody responses of 36 children with symptomatic malaria to a diverse panel of 36 recombinant proteins comprising part of the DBLα domain (the 'DBLα-tag') of PfEMP1, a major class of VSAs. We found that although plasma antibody responses were highly specific to individual antigens, serological profiles of responses across antigens fell into one of just two distinct types. One type was found almost exclusively in children that succumbed to severe disease (19 out of 20) while the other occurred in all children with mild disease (16 out of 16). Moreover, children with severe malaria had serological profiles that were narrower in antigen specificity and shorter-lived than those in children with mild malaria. Borrowing a novel technique used in influenza-antigenic cartography-we mapped these dichotomous serological profiles to amino acid sequence variation within a small sub-region of the PfEMP1 DBLα domain. By applying our methodology on a larger scale, it should be possible to identify epitopes responsible for eliciting the protective version of serological profiles to PfEMP1 thereby accelerating development of a broadly effective anti-disease malaria vaccine.

The effect of fatty acids in red blood cell membranes on the dynamics of inflammatory markers following the coronary stent implantation.

The effect of 20 fatty acids in erythrocyte cell membranes on the extent of inflammatory response and cell oxidative stress was evaluated using multidimensional statistical data analysis in 54 patients suffering from ischemic heart disease undergoing percutaneous coronary intervention with coronary stent implantation using multidimensional statistical data analysis. A systemic inflammatory response was indicated by an increase of C-reactive protein (CRP), serum amyloid A (SAA) and ceruloplasmin 48 h after stent implantation and by an increase of interleukin-6 (IL-6) 24 h after intervention. The increase of malondialdehyde (MDA) after 48 h was used as a marker of cell damage by oxidative stress. Multiple linear regression revealed statistically significant relationships between concentration of some fatty acids and the magnitude of inflammatory response, or oxidative stress, after stent implantation. The most significant relationship with an increase of plasma CRP was found for myristic acid and, to a lesser extent, for oleic acid. Trans octadecenoic acid, and to a lesser extent palmitooleic and nervonic fatty acids were found in inverse correlation with the CRP increase. The increase of IL-6 showed a statistically significant correlation with myristic acid, to a lesser extent with cis-9-eicosenoic acid and to the least extent with docosahexaenoic acid, inversely with pentadecanoic, γ-linolenic and stearic acids. An increase of oxidative stress (MDA) significantly correlated only with γ-linolenic acid. Other studied markers of inflammatory response to coronary stenting were SAA and ceruloplasmin (Cp). Statistical evaluation revealed that SAA and Cp are not suitable markers for assessment relationships between inflammation and erythrocyte membrane fatty acids.

Development of RBC Membrane Antigen Arrays for Validating Blood Grouping Reagents.

Antibody reagents have been remained as a standard approach to characterize blood group (BG) antigens in clinic. The specificity and cross-reactivity of these BG antibodies are routine detected using the gel microcolumn assay (GMA). However, the GMA is neither specific nor sensitive, thus increasing the risk of improperly matched RBC transfusions. In this work, we describe a bead-based RBC membrane antigen array to detect BG antibody-antigen binding with ∼700-fold higher sensitivity and dynamic range than the GMA. RBC membrane antigen arrays were fabricated using fragmented RBC membranes highly enriched in BG panel antigens. The arrays were then used to screen the interactions of 15 BG reagents to three antigen panels. The majority of the antibody reactions (i.e., 86.7%; 39/45) aligned with those obtained with the GMA. The six cross-reactive, nonspecific antibody reactions identified only by our arrays (i.e., 13.3%; 6/45) were confirmed by agglutination inhibition and genotyping assays. These results demonstrate that our RBC membrane antigen array has great potential in screening BG antibodies and improving the safety of RBC transfusions.

Daratumumab: Therapeutic asset, biological trap!

OBJECTIVES: Recently, daratumumab has been included in the therapeutic strategies for myeloma patients. This molecule is an antibody directed against CD38, strongly expressed on plasma cells. Nevertheless, as CD38 is also present on erythrocyte membrane, daratumumab interferes with immunohaematological tests, complicating the selection of compatible blood. METHODS: A total of 14 patients treated by daratumumab have been followed in our transfusion laboratory. Among them, 11 have been transfused. Dithiotreitol (DTT) has been used to inhibit the daratumumab's interference, in the pre-transfusion tests (irregular antibody screening and cross-match). RESULTS: The red blood cell treatment with DTT has been very efficacious to inhibit the daratumumab's interference in 13 patients out of 14. Some precautionary measures had to be taken into account, especially the pH and the storage conditions. An extended pheno/genotype was an additional security element in the selection of compatible blood. To simplify and to optimize the laboratory practices, a decisional flow chart has been written. CONCLUSION: DTT red blood cell treatment is very useful and efficacious in the pre-transfusion tests of patients treated with daratumumab. It allows to avoid the selection of blood bags only on the basis of an extended pheno/genotype, what is more secure and more ethical with respect to other at higher risk patients. A clear decisional flow chart allows a quality assurance gait. Collaboration with physicians is essential.

The immune-stimulation capacity of liposome-treated red blood cells.

Our in vivo studies on a rat model established safety of transfusing liposome-treated red blood cells (RBCs) but identified the potential for immune modulation as related to transfusion efficacy of liposome-treated RBCs. The aim of this study was at assessing the impact of liposome-induced membrane changes on the immune profile of liposome-treated RBCs by (a) evaluating their interaction with endothelial cells and monocytes; and (b) the resulting immune response derived from this interaction, in the form of cytokine release, adhesion molecules expression and phagocytosis. Unilamellar liposomes were synthesized to contain unsaturated phospholipids (1,2-dioleoyl-sn-glycero-3-phosphocholine [DOPC]:CHOL, 7:3 mol%). The human RBCs immune profile was assessed by incubating control and DOPC-treated RBCs with human umbilical vein endothelial cells (HUVECs) and monocytes. Cytokine release measured by Luminex technology, vascular cell adhesion molecule (VCAM)-1 and E-selectin on HUVECs measured by flow cytometry, and the erythrophagocytic activity of monocytes by monocyte monolayer assay (MMA) were determined. Fibroblast growth factor [FGF]-2 was the only cytokine released by HUVECs that remained increased after incubation with DOPC-treated RBCs compared to control throughout storage. The expression of both VCAM-1 (15.3 ± 5.6% versus 6.3 ± 0.9%, p = 0.008) and E-selectin (18.0 ± 6.3% versus 6.6 ± 0.7%, p = 0.004) by HUVECs were significantly increased after incubation with DOPC-treated RBCs at day 2 of storage. The MMA resulted in phagocytic indexes of zero for both control and DOPC-treated RBCs at day 2 and 42 of storage. The liposome treatment did not result in significant changes to the immune profile of stored DOPC-treated RBCs. These findings combined with previous in vivo results, make liposome treatment a potential candidate for application in RBC preservation and open the possibility for clinical use with other cell types.

[Is the research of posttransfusional alloantibodies still relevant?] / Est-ce que la réalisation de la recherche d'anticorps antiérythrocytaires post­transfusionnelle est toujours pertinente ?

The decision of November 6th, 2006 defining the principles of best practices recommends that posttransfusional red cell alloantibodies research is performed after one to three months after. In the University hospital of Brest, the haemovigilance unit takes charge of sending the medical prescription within the required time and centralizing the results. We wished to estimate if the realization of this research still remains relevant. METHODS: A prospective analysis was performed in 2015. We evaluated the realization rate, the red cell alloantibodies rate and the recipient adverse reactions with the diagnostic category: alloimmunization (delayed serological transfusion reaction, DSTR). RESULTS: In 2015, 2162 prescriptions were sent to the 3271 transfused patients. One thousand and eighteen red cell alloantibodies research were done, i.e. a return rate of 61%. Among them, 12 alloantibodies appeared (0.9%) within an average of 56 days. Thirty-three other alloantibodies appeared and were discovered most frequently before a new transfusion. In 10 cases, a posttransfusional research was done that was negative. A survey was conducted among GHCOH members to describe the practices in these health institutions. Twelve questionnaires were analysed. Ten institutions performed a posttransfusional alloantibodies research by issuing a prescription at the patient's exit with a return rate between 0.14 and 16%; 1 institution has a centralized organization with a return rate of 68.3%; 1566 red cell alloantibodies research were performed and among them, 24 alloantibodies appeared (1.53%). CONCLUSION: These results indicate that to be effective, the management of this biological test must be centralized. Despite this, the red cell alloantibodies rate remains very low (0.9 and 1.53%) and raises the question of the relevance of this systematic testing after transfusion, which is in any case mandatory before a new transfusion of red blood cells.

Immunoproteomics of Plasmodium falciparum-infected red blood cell membrane fractions.

BACKGROUND: The surface of infected red blood cells (iRBCs) has been widely investigated because of the molecular complexity and pathogenesis mechanisms involved. Asymptomatic individuals are important in the field because they can perpetuate transmission as natural reservoirs and present a challenge for diagnosing malaria because of their low levels of circulating parasites. Recent studies of iRBC antibody recognition have shown that responses are quantitatively similar in symptomatic and asymptomatic infections, but no studies have characterised the plasmodial proteins targeted by this response. OBJECTIVES: Our main objective was to identify Plasmodium falciparum proteins associated with iRBC ghosts recognised by antibodies in the sera of symptomatic and asymptomatic individuals in the Brazilian Amazon. METHODS: We collected symptomatic and asymptomatic sera from patients residing in the Brazilian Amazon and P. falciparum iRBC ghosts to identify the proteins involved in natural antibody recognition by 2D-electrophoresis, western blotting, and high- resolution mass spectrometry. FINDINGS: 2D gel-based immunoproteome analysis using symptomatic and asymptomatic sera identified 11 proteins with at least one unique peptide, such as chaperones HSP70-1 and HSP70-x, which likely are components of the secretion machinery/PTEX translocon. PfEMP1 is involved in antigenic variation in symptomatic infections and we found putative membrane proteins whose functions are unknown. MAIN FINDINGS: Our results suggest a potential role of old and new proteins, such as antigenic variation proteins, iRBC remodelling, and membrane proteins, with no assigned functions related to the immune response against P. falciparum, providing insights into the pathogenesis, erythrocyte remodelling, and secretion machinery important for alternative diagnosis and/or malaria therapy.

Immunoproteomics of Plasmodium falciparum-infected red blood cell membrane fractions

BACKGROUND The surface of infected red blood cells (iRBCs) has been widely investigated because of the molecular complexity and pathogenesis mechanisms involved. Asymptomatic individuals are important in the field because they can perpetuate transmission as natural reservoirs and present a challenge for diagnosing malaria because of their low levels of circulating parasites. Recent studies of iRBC antibody recognition have shown that responses are quantitatively similar in symptomatic and asymptomatic infections, but no studies have characterised the plasmodial proteins targeted by this response. OBJECTIVES Our main objective was to identify Plasmodium falciparum proteins associated with iRBC ghosts recognised by antibodies in the sera of symptomatic and asymptomatic individuals in the Brazilian Amazon. METHODS We collected symptomatic and asymptomatic sera from patients residing in the Brazilian Amazon and P. falciparum iRBC ghosts to identify the proteins involved in natural antibody recognition by 2D-electrophoresis, western blotting, and high- resolution mass spectrometry. FINDINGS 2D gel-based immunoproteome analysis using symptomatic and asymptomatic sera identified 11 proteins with at least one unique peptide, such as chaperones HSP70-1 and HSP70-x, which likely are components of the secretion machinery/PTEX translocon. PfEMP1 is involved in antigenic variation in symptomatic infections and we found putative membrane proteins whose functions are unknown. MAIN FINDINGS Our results suggest a potential role of old and new proteins, such as antigenic variation proteins, iRBC remodelling, and membrane proteins, with no assigned functions related to the immune response against P. falciparum, providing insights into the pathogenesis, erythrocyte remodelling, and secretion machinery important for alternative diagnosis and/or malaria therapy.